Sample injector for high pressure liquid chromatography

ABSTRACT

Apparatus and method for driving a sample, having a well-defined volume, under pressure into a chromatography column. A conventional high pressure sampling valve is replaced by a sample injector composed of a pair of injector components connected in series to a common junction. The injector components are constructed so as to provide for electroosmotic flow of a sample into the junction. At an appropriate time, a pressure pulse from a high pressure source, that can be an electrokinetic pump, connected to the common junction, drives a portion of the sample, whose size is determined by the dead volume of the common junction, into the chromatographic column for subsequent separation and analysis. The apparatus can be fabricated on a substrate for microanalytical applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Not applicable.

STATEMENT OF GOVERNMENT INTEREST

[0002] This invention was made with Government support under contractno. DE-AC04-94AL85000 awarded by the U. S. Department of Energy toSandia Corporation. The Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] This invention is directed to an apparatus for high pressureinjection of a sample into chromatography apparatus generally and intohigh pressure liquid chromatographic (HPLC) apparatus in particular. Thedesign of this apparatus is such that the phenomenon of sample “tailing”is substantially eliminated. Thus, the apparatus is particularly usefulfor capillary-based chromatographic systems.

[0004] A typical prior art method of injecting a sample intopressure-driven chromatography systems, such as an HPLC apparatus, isillustrated schematically in FIG. 1. Here, a source of pressure, whichin conventional chromatography systems is generally a piston orcam-driven pump, is initially, used to force a stream of buffer solutionthrough a packed HPLC column. At the proper time, a sampling valve isturned admitting a portion of a sample into the flowing buffer solutionstream and onto the HPLC column where the various components areseparated and pass to a detector for analysis. However, for accurateanalysis, particularly for complex samples, it is necessary to controlprecisely the opening and closing of the sampling valve to minimizeflow-induced mixing and dead volume, all of which are very difficult todo.

[0005] Miniaturization of the chromatography apparatus offers severaladvantages including, improved efficiency, greater detectionsensitivity, low solvent consumption, speed, and the need for only smallquantities of sample (typically in the μL range). In the extreme,complete microscale chromatography systems have been developed that fitinto a single cm-size substrate. Examples of these systems can be foundin U.S. Pat. No. 5,885,470 to Parce et al., U.S. Pat. No. 5,858,195 andInternational Application WO 96/04547 to Ramsey, and U.S. Pat. No.5,571,410 to Swedberg et al. However, in microanalytical pressure-drivenchromatography systems the problem of providing a sample having awell-defined volume is exacerbated. It has been found that it isextremely difficult to define accurately a sample volume injected intothe chromatography column since the sample itself is quite small. Thisis generally a consequence of the fact that sampling valves suitable forpressure-driven microscale chromatography systems are either notavailable or are incapable of being opened and closed precisely enoughto eliminate sample “tailing”, flow-induced mixing and dead volume.Moreover, there can be changes in sample composition since fastermigrating compounds will be introduced into the chromatography columnpreferentially. There have been many attempts to alleviate sample“tailing” problem inherent in pressure-driven microanalyticalchromatography systems, none have been entirely successful.

SUMMARY OF THE INVENTION

[0006] It is an object of this invention to provide an apparatus thatprovides a clean injection, i.e., sample injection with substantially notailing, of a sample into pressure-driven chromatography systems.

[0007] It is a further object of this invention to provide an apparatusfor injecting a sample into an HPLC system that substantially eliminatessample tailing.

[0008] It is yet another object of this invention to provide anapparatus for sample injection into a microanalytical HPLC system.

[0009] Another object of the invention is to provide a method for sampleinjection that yields a well-defined sample volume.

[0010] A further object is to provide a method for sample injection intomicroanalytical chromatography systems.

[0011] Sample injection into a chromatography system, and particularlyinto an HPLC system, is accomplished by the present invention by meansof electroosmotic/electrophoretic (EO/EP) injection. Here, aconventional sampling valve, such as that illustrated in FIG. 1, isreplaced by a pair of injector elements connected in series to a commonjunction disposed at the inlet to a chromatography column. Theseelements initiate sample flow through the common junction. At anappropriate time, a pressure pulse from a high pressure pumping system,such as an electrokinetic pump (EKP) drives that portion of the sampleresiding in the volume of the common junction, and whose size isdetermined by the volume of the common junction, into the chromatographycolumn. The advantage offered by this means of sample injection is thatapplication of the pressure pulse that drives the sample into thechromatography column eliminates sample leakage from the injectorelements thereby providing a well-defined sample plug, i.e., a samplethat exhibits substantially no tailing.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 illustrates a conventional sample injection method.

[0013]FIG. 2 illustrates an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention is directed to method and apparatus forinjecting a sample having a well-defined volume into pressure-drivenchromatography systems, and particularly into high pressure liquidchromatography (HPLC) systems. The apparatus provides a substantially“clean” injection, i.e., a sample plug having little or no “tailing”produced by leakage of sample from injector elements subsequent to thestep of injection, and thus finds particular application inmicroanalytical chromatography systems.

[0015] The present invention is illustrated and exemplified by theembodiment shown in FIG. 2. Here, a source of hydrostatic pressure 310is joined at a common junction 335 to an HPLC column 320 to supply arunning buffer or other chromatographic fluid 315 to the column. Asillustrated in FIG. 2, a preferred hydrostatic pressure source is anelectrokinetic pump (EKP). By converting electric potential tohydrodynamic force, an EKP is capable of exerting hydrostatic pressuresin excess of 10,000 psi. Moreover, in contrast to prior art pumps, anEKP has no moving mechanical parts and control of pressure and solventflowrate is achieved simply by controlling the voltage applied to theEKP by voltage source 330. The EKP and its operation has been fullydescribed in U.S. Pat. No. 6,103,164, issued Jan. 11, 2000 and entitled“Electrokinetic High Pressure Hydraulic System” and is incorporatedherein in its entirety.

[0016] It will be appreciated by those skilled in the art that it isdesirable to eliminate the generation of any gases that could arise as aconsequence of electrolysis of the EKP electrolyte. This can beaccomplished by means known to the art. By way of example, a section ofultra micro-porous material, such as the porous glass sold under thetrademark VYCOR, having nominally 4 nm pores, or a membrane such as thatsold under the trademark NAFION, saturated with electrolyte can beinterposed between the electrode providing connection to the highpressure fluid junction and the junction itself 380. The ultramicro-porous material carries current but the pores are sufficientlyfine so that transport of material by pressure-driven or electroosmoticflow is negligible.

[0017] A sample injector A is also connected to common junction 335between hydrostatic pressure source 310 and HPLC column 320. Theinjector is comprised of at least two elements such as 340 and 350. Eachelement comprises a container having an inlet and outlet end and filledwith a dielectric material to form a porous bed within the container.Containers can include any geometric configuration capable of containingthe porous bed of dielectric material, such as capillary tubes, andcapable of withstanding pressures of up to about 40,000 psi. Alsoincluded are microchannels fabricated on a substrate such as thosedescribed by Paul et al. in U.S. Pat. Nos. 6,103,104 and 6,019,882 andby Arnold in prior co-pending U.S. patent application Ser. No.09/404,945, filed Sep. 23, 1999, entitled “Microfluidic ChannelFabrication Method” assigned to the same assignee. Elements 340 and 350are connected together in series configuration with a common junction335. The dielectric material filling each container is selected so as tominimize any chromatographic separation of the sample and can be anynon-porous material, known to those skilled in the art, used to form aporous, packed bed. By way of example, the dielectric material can becomprised of uncoated and nonporous silica, glass, or polymer beads or aporous monolithic polymer material. Further, the dielectric material isselected to resist pressure-driven flow but to allowelectroosmotically-driven flow. Thus, additionally it is preferred thatthe pore diameter of the porous bed be in the range of about 25 to 300nm. One of elements 340/350 serves as a sample inlet and is incommunication with a sample container 360 and the other is connected toa waste reservoir 370. A power supply 375 is connected across bothelements of sample injector A. Placing one of the electrodes of powersupply 375 in the sample container and the other in the waste reservoircan make this connection. Alternatively, power supply 375 can beconnected to the elements of the sample injector by means of saltbridges, as discussed above.

[0018] One method of injecting a sample into HPLC column 320 can be torun EKP 310 to flush HPLC column 320 with a clean running buffersolution. At an appropriate time, the voltage to EKP 310 is shut off byopening switch 390 and a voltage applied to elements 340 and 350 ofsample injector A by closing switch 385, thereby causing liquid to flowfrom sample container 360 through sample injector A. After a flow of thesample to be analyzed has been established through junction 335, switch385 is opened to shut off voltage supplied to injector elements 340 and350. At this point, voltage is once again supplied to EKP 310 therebycausing that portion of the sample that is resident in common junction335 to be pressure-driven into HPLC column 320 by the running buffer,the volume of the sample being determined by the dead volume of thecommon junction. As the sample is driven through the HPLC column by therunning buffer it is separated into its components, which are detectedat the HPLC column outlet.

[0019] It should be noted that because the porous bed of dielectricmaterial contained in each of the components of the sample injector hasa pore size of about 35 to 300 nm, the bed presents a very highresistance to pressure-driven flow. Thus, these components themselvescan act as a form of check valve when, having achieved a high pressure,the electric potential supplied to them is switched off.

[0020] The present invention now makes it possible to fabricate acompletely pressure-driven microanalytical chromatography system on asubstrate. Heretofore, because sampling valves suitable for microscalechromatography systems either were not available or were incapable ofbeing opened and/or closed precisely enough to eliminate sample“tailing”, flow-induced mixing and dead volume, pressure-drivenmicroanalytical chromatography systems were precluded. Thus, in atypical microanalytical chromatography systems samples are moved byelectroosmotic force, wherein an electric field is applied to cause thesample to move throughout the system in a series of interconnectedchannels such as those described by Ramsey. However, the use ofelectroosmotic force to move samples can cause changes in samplecomposition since faster migrating compounds will be introduced into thechromatography column preferentially. This source of error inmicroanalytical systems is eliminated by the present invention by theuse of pressure-driven sample injection.

[0021] It should be noted that in addition to the dielectric materialsset forth above that can be placed into a previously fabricated channeldisposed on a substrate to comprise the porous bed, other high surfacearea features such as might be produced by microfabrication methodsknown to those skilled in the art, preferably by lithographic etching,and which present a porous matrix having a high surface area can bemicrofabricated directly into the channel.

[0022] It will be understood that the described arrangement of apparatusand methods pertaining thereto are merely illustrative of applicationsof the principles of this and many other embodiments and modificationscan be made by those of skill in the art without departing from thespirit and scope of the invention as defined in the claims.

We claim:
 1. An apparatus for injecting a sample into a chromatographiccolumn, comprising: a source of high pressure joined at a commonjunction to the inlet of the chromatographic column; and a sampleinjector joined to the common junction, said sample injector comprisingat least two elements combined in a series arrangement and connected toa common power supply, wherein each element comprises a container havingan inlet end and an outlet end, the container filled with a dielectricmaterial forming a porous bed therein.
 2. The apparatus of claim 1 ,wherein said high pressure source comprises an electrokinetic pump. 3.The apparatus of claim 1 , wherein the porous dielectric material iscomprised of a nonporous and uncoated material.
 4. The apparatus ofclaim 3 , wherein the dielectric material includes a glass, ceramic, orpolymer material.
 5. The apparatus of claim 4 , wherein the dielectricmaterial is silica beads.
 6. The apparatus of claim 1 , wherein theporous bed has a pore size in the range of about 25 to 300 nm.
 7. Theapparatus of claim 1 , further including at least one salt bridgedisposed between the electrodes of the power supply and the elements ofsaid sample injector.
 8. A method of injecting a sample into achromatography column for chromatographic analysis, comprising the stepsof: providing a flow of a running buffer to the chromatography column;shutting off the flow of running buffer; applying a voltage to a sampleinjector having a common junction with the chromatography column tocause a sample to flow into the common junction; and starting the flowof running buffer to apply a pressure to drive the sample contained inthe common junction into and through the chromatography column.
 9. Anapparatus for the high pressure injection of a sample into achromatography column, comprising: a substrate fabricated to define amicrochannel system disposed thereon, the microchannel systemcomprising, in combination, a source of high pressure joined at a commonjunction to the inlet of the chromatography column; and a sampleinjector joined to the to the common junction, said sample injectorcomprising at least two elements combined in a series arrangement andconnected to a common power supply, wherein each element comprises acontainer having an inlet end and an outlet end, the container filledwith a dielectric material forming a porous bed therein.
 10. Theapparatus of claim 9 , wherein the source of high pressure is anelectrokinetic pump.
 11. The apparatus of claim 9 , further including atleast one salt bridge disposed between the electrodes of the powersupply and the elements of said sample injector.
 12. The apparatus ofclaim 9 , wherein the dielectric material comprising the porous bed isproduced by lithographic etching.